Coupling Of Dielectrophoresis And Spatial Effects In Microfluidic Chip For Circulating Tumor Cell Sorting

Cell sorting is widely applied in the field of biology or clinical medicine.Compared with the traditional flow cytometric sorting method,microfluidic cell sorting has become a research hotspot in cell sorting field with many advantages of high throughput,rapid and easy operation and availability to combine autonomously.Among the classical active microfluidic cell sorting methods,dielectrophoresis sorting does not require labeling cells,but merely relies on the physical properties of the cell namely dielectric properties and cell size,and it is convenient to operate and can reach high sorting efficiency.But its application is limited to sorting cells with large differences in dielectric properties.This thesis continues the research work of our group on microfluidic dielectrophoresis cell sorting.Considering the former dielectrophoresis sorting chip we made or single dielectrophoresis sorting method is unable to well separate cells with similar dielectric properties,a new type of chip is designed based on the spatial effect in complicated microchannel,which introduces hydrodynamic units in channel design and couples the dielectrophoresis sorting with hydrodynamic sorting to realize the synergistic effect of these two acting forces and achieve the purpose of separating cancer cells from blood cells.First of all,soft lithography was used to construct a variety of PDMS microchannels with spatial sorting effect.The trials show that contraction/expansion structure has the best sorting effect by comparing the cell sorting effect.Meanwhile 3D sidewall electrodes(thickness about 25 ?m)were prepared by screen printing method.The problems occurred in the screen printing process were studied in detail and corresponding solutions were proposed.Based on the relative positions of the 3D sidewall electrodes and the contraction/expansion structure,three types of microchips(full-coupling,semi-coupling,non-coupling)were constructed.All of them have 12 contraction/expansion units and the same electrode length,for ensuring the equal time of hydrodynamics functioned and dielectrophoresis functioned.Next,as a practical example of sorting rare cells such as circulating tumor cells from the blood,we selected rabbit red blood cells(8 ?m in diameter)and lung cancer cells PC-9(16 ?m in diameter)and mixed them in a ratio of 1000:1.Comsol software was used to simulate the sorting performance of three kinds of coupling chips on this cell system.The electric field distribution and fluid field distribution in the chip were analyzed by finite element simulation with multi-physics modules.Based on this calculation,the particle tracking module was used to predict the cell trajectories during sorting.The simulation results show that the full-coupling chip has the best sorting performance.And then,Dielectric spectra of PC-9 cells and red blood cells were obtained based on static dielectric properties experiments.A sorting platform was set up where sorting experiments were performed within three kinds of chips.The experimental results also showed that full-coupling microchips can achieve higher cell sorting efficiency(PC-9 cell recovery: 90.21%,RBC recovery: 94.35%).To explore the mechanism of sorting on full-coupling chips,sorting experiments were respectively performed on pure contraction/expansion chip,parallel-electrode structured chips,and full-coupling chip,hence a new sorting mechanism was proposed by comparing sorting process: the synergy of DEP sorting and hydrodynamic sorting promotes effective cell sorting.That is,the positive dielectric force generated by the 3D sidewall electrodes can simultaneously act as an additional shear gradient lift force to induce secondary flow at low flow rates.Finally,the conditions for cell sorting within full-coupling chip were optimized,and the effects of medium conductivity,flow rate,input voltage,and input frequency on sorting efficiency were explored.The results showed that sorting efficiency was optimal on the condition that flow rate is 15?L/h,medium conductivity 80 ?S/,input voltage 13 V,input frequency 900 kHZ.Through a series of live-dyed staining,hemolysis assay,and CCK-8 assay,the effect of sorting process on cell activity,morphology and proliferation was evaluated.It turned out there was no significant difference in the biological behavior of red blood cells and PC-9 cells before and after sorting.In summary,our results show that the microfluidic chip coupled with dielectrophoresis unit and hydrodynamic unit can achieve the sorting of circulating tumor cells in the blood,and this work also show the proposed coupling platform brings on a new pathway to construct integrated microfluidic chips of multiple cell sorting methods for cell sorting and separation.